The proposed research is a biochemical-genetic study of mitochondrial rRNA splicing in Neurospora. Recent studies have shown that the gene encoding the 25 S (large subunit) mitochondrial rRNA of Neurospora contains an intervening sequence of ca. 2.3 kb. We have identified two temperature-sensitive, nuclear mutants which are defective in splicing this RNA. When grown at the nonpermissive temperature, the mutants accumulate a 35 S precursor RNA which appears to be a continuous transcript of the 25 S RNA gene including the intervening sequence. Specific aims are: (a) to continue to characterize the structure of the 35 S RNA, (b) to use the 35 S RNA as substrate to assay and isolate the RNS splicing enzymes and (c) to continue to isolate and characterize revertants of the mutants. These studies are intended to provide insight into the mechanism and physiological role of RNA splicing which should be relevant to all eukaryotic cells.